Emerging infectious diseases together with the intentional use of pathogenic organisms or their toxins for acts of terror constitute some of the most significant health and security challenges facing the global community. A key challenge is to develop effective strategies to rapidly detect and control naturally occurring outbreaks of infectious disease. Especially for biodefense and risk evaluation, rapid and definitive identification of pathogens and their toxins is of enormous significance. Real-time, multiplexing capabilities are essential in any diagnostic for surveillance, confirmation, and timely implementation of preventive and protective public health measures; however, conventional diagnostics fail to provide these capabilities in a format that allows for deployment in limited resource, point-of-care settings. Consequently, there is a critical need for robust multiplex strategies to identify pathogens and toxins that can be readily deployed in virtually any environmental and clinical setting and easily configured (e.g., targeted for geographic pathogen serogroups and human populations for which it is intended). To address these issues, this project proposes to fabricate and establish proof-of-concept of a prototype next generation, multiplex diagnostic for detection of select biodefense and emerging pathogens. The innovative sensor design will constitute a highly ordered array of discrete, electrically addressable nanocoaxial sensors arrays, each with a specified molecular imprint (MI) nanocoating for recognition of a distinct toxin or pathogen. This novel sensor array will enable: 1) multiplexing and proofreading capability, due to its molecular scale and architecture that allows for spatially discrete, high site density (~108 per cm2), electrically addressable nanocoaxial sensors; 2) unprecedented sensitivity and specificity; and 3) all-electronic capacitance measurement that affords label-free detection, ideally suited for limited resource, field-appropriate settings on a scale that has not previously been achievable. The R21 component will fabricate nanocoaxial sensors containing MIs capable of ultrasensitive and specific toxin recognition and will establish proof-of-concept for the simultaneous detection of cholera and shiga toxins using nanocoaxial sensor arrays on a single multiplex chip. Expanding on these results, the R33 component will seek to evaluate proof-of-concept for detection of pathogenic organisms by molecular imprinted nanocoaxial sensor arrays, using Vibrio cholerae strains belong to the O1 and O139 serogroups and Shiga pathogen-producing Escherichia coli (STEC) O157:H7. The R33 component will also engineer sample delivery and integrated electronics into a prototype handheld diagnostic for simultaneous detection of both pathogens and toxins on a single disposable multiplex chip. PUBLIC HEALTH RELEVANCE: Emerging infectious diseases together with the intentional use of pathogenic organisms or their toxins for acts of terror constitute some of the most significant health and security challenges facing the United States and the global community. Consequently, there is a critical need for robust diagnostics to identify pathogens and toxins that can be deployed in virtually any environmental and clinical setting, and easily configured for distinct geographic pathogen serogroups and human populations for which it is intended. To address these issues, this project proposes to fabricate and establish proof-of-concept of a prototype next generation, multiplex nanoscale diagnostic device that can be deployed in limited resource, point-of-care settings, for detection of select biodefense and emerging pathogens.